Project Summary The abundance of crystal structures determined for kinase domains of human receptor tyrosine kinases (RTKs) deposited in the protein data bank have provided snapshots of these enzymes in both inactive and active states. However, our knowledge of dynamics of this highly allosteric class of enzymes is still in its infancy. The overall goal of this proposal is to elucidate the intrinsic and extrinsic allosteric control mechanisms that underlie tyrosine kinase regulation by using the fibroblast growth factor receptor (FGFR) kinase subfamily as the model system. FGF signaling fulfills a multitude of diverse biological functions throughout embryonic development and adulthood by controlling cellular proliferation, differentiation, chemotaxis, apoptosis, and senescence. Through concerted crystallographic and NMR solution studies of a large set of gain-of-function mutations, we have recently elucidated a dynamic two-state model for FGFR kinase regulation whereby the enzyme toggles between an inhibited, conformationally rigid state and a more flexible active state (Molecular Cell, 2007; Cell Reports, 2013). More recently we have refined this model to show that the concerted action of four molecular switches forming a long-range allosteric connectivity stretching from the kinase hinge located at the back of the kinase all the way to the A-loop and catalytic pocket at the front of the kinase regulate the dynamics and thus the active-inactive equilibrium. These data have provided a solid basis for tackling imminent problems in our comprehension of RTK signaling that are carefully formulated in each of three aims of this proposal. In Aim I, we will demonstrate for the first time how differences in intrinsic dynamics of four human FGFR isoforms account for their distinct signaling potentials, thereby providing a molecular rationale for the evolution of multimember RTK subfamilies. In Aim II, we will determine how frequently occurring mutations at the gate-keeper residue, a major hurdle in the clinic for drug-resistance, corrupts the autoinhibitory interactions and leads to gain-of-function. In Aim III, we will establish the structural and dynamic basis by which FGFR recruits and phosphorylates its major intracellular substrate, FRS2? and demonstrate for the first time how intracellular substrate binding can act as an extrinsic factor to regulate intrinsic kinase activity. By using a hybrid of structural experiments (X-ray crystallography and NMR spectroscopy) and biological assays (in vitro and in cells), we will accomplish each of the aims described that represent major milestones in the RTK field. This research will fill several knowledge gaps in our understanding of RTK signaling and hence will have a powerful and sustained influence in the cellular signaling field.